Diet-induced obesity affects influenza disease severity and transmission dynamics in ferrets

Obesity, and the associated metabolic syndrome, is a risk factor for increased disease severity with a variety of infectious agents, including influenza virus. Yet, the mechanisms are only partially understood. As the number of people, particularly children, living with obesity continues to rise, it is critical to understand the role of host status on disease pathogenesis. In these studies, we use a diet-induced obese ferret model and tools to demonstrate that, like humans, obesity resulted in notable changes to the lung microenvironment, leading to increased clinical disease and viral spread to the lower respiratory tract. The decreased antiviral responses also resulted in obese animals shedding higher infectious virus for a longer period, making them more likely to transmit to contacts. These data suggest that the obese ferret model may be crucial to understanding obesity’s impact on influenza disease severity and community transmission and a key tool for therapeutic and intervention development for this high-risk population.


Fig. S2 .
Fig. S2.Individual growth kinetics of ferrets on diet protocol.Male ferrets (n=40 total) placed on diet protocol with individual ferret growth recorded as (A) weight measured in kilograms, (B) waist circumference measured in centimeters, (C) skinfold fat measured in millimeters, and (D) ferret mass index measured by standardizing the product of weight in kilograms and circumference in centimeters by the ferret length in centimeters squared.Each column is representative of one ferret measured weekly for 12 weeks.Individual data underlies average values plotted in Fig. S1.

Fig. S3 .
Fig. S3.Symptom log of H1N1 virus-inoculated ferrets.Obese (n=10) or control (n=14) ferrets were intranasally inoculated with 10 6 TCID50 of A/California/04/2009 (H1N1) influenza virus and monitored for 12 days post-infection.Severity of (A) temperature, (B) weight change, and (C) sum symptoms are recorded for each ferret.Sum symptom score comprises (D) activity level, (E) severity of sneezing, (F) nasal wash grade, (G) presence of nasal discharge, (H) presence of coughing, and (I) presence of conjunctivitis.This data corresponds with Fig. 2C.Symptoms were recorded daily for (A-B, D-E, and G-I) and every other day for (C, F) due to nasal wash sampling schedule.Historically, ferrets display none-to-mild symptoms during infection with human influenza viruses so the literature reports weights and temperatures; however, due to the more severe symptoms observed in the obese ferrets we expanded our clinical scoring scale.Therefore (D-I) are reported for only n=6 obese and n=6 or 8 control ferrets per diet group.

Fig. S5 .
Fig. S5.Symptom log of H3N2 virus-inoculated ferrets.Obese (n=10) or control (n=11) ferrets were intranasally inoculated with 10 6 TCID50 of A/Memphis/257/2019 (H3N2) influenza virus and monitored for 12 days post-infection.Severity of (A) temperature, (B) weight change, and (C) sum symptoms are recorded for each ferret.Sum symptom score comprises (D) activity level, (E) severity of sneezing, (F) nasal wash grade, (G) presence of nasal discharge, (H) presence of coughing, and (I) presence of conjunctivitis.This data corresponds with Fig. 4A, D, G. Symptoms were recorded daily for (A-B, D-E, and G-I) and every other day for (C, F) due to nasal wash sampling schedule.

Fig. S6 .
Fig. S6.Symptom log of H9N2 virus-inoculated ferrets.Obese (n=17) or control (n=10) ferrets were intranasally inoculated with 10 6 TCID50 of A/Hong Kong/1079/1999 (H9N2) influenza virus and monitored for 12 days post-infection.Severity of (A) temperature, (B) weight change, and (C) sum symptoms are recorded for each ferret.Sum symptom score comprises (D) activity level, (E) severity of sneezing, (F) nasal wash grade, (G) presence of nasal discharge, (H) presence of coughing, and (I) presence of conjunctivitis.These data correspond with Fig. 4B, E, H. Symptoms were recorded daily for (A-B, D-E, and G-I) and every other day for (C, F) due to nasal wash sampling schedule.Historically, ferrets display none-to-mild symptoms during infection with human influenza viruses so the literature reports weights and temperatures; however, due to the more severe symptoms observed in the obese ferrets we expanded our clinical scoring scale.Therefore (C-I) are reported for only n=5 obese and n=3 control ferrets.(J) Hematoxylin and eosin staining of lungs taken from ferrets infected with 10 6 TCID50 A/Hong Kong/1079/1999 (H9N2) virus at 6 dpi.Data represents 2 independent experiments of n=2/group with 5 histology

Fig. S7 .
Fig. S7.Symptom log of influenza B virus-inoculated ferrets.Obese (n=8) or control (n=8) ferrets were intranasally inoculated with 10 5.5 TCID50 of B/Brisbane/60/2008 influenza virus and monitored for 12 days post-infection.Severity of (A) temperature, (B) weight change, and (C) sum symptoms are recorded for each ferret.Sum symptom score comprises (D) activity level, (E) severity of sneezing, (F) nasal wash grade, (G) presence of nasal discharge, (H) presence of coughing, and (I) presence of conjunctivitis.This data corresponds with Fig. 4C, F, I. Symptoms were recorded daily for (A-B, D-E, and G-I) and every other day for (C, F) due to nasal wash sampling schedule.